In the field of stable isotope measurements for medical diagnostics, the rate of change of the isotopic ratios is used to study energy expenditure, metabolic studies, and glucose metabolism. In order to analyze a blood sample, it must first be fractionated into blood plasma (approx. 55%) and cellular material (i.e., erythrocytes, leukocytes and platelets, approx. 45%), usually by centrifugation, and then the extracted blood plasma must be further processed so as to remove the albumin, fibrinogen, immunoglobulin and other proteins that would otherwise tend to clog the analysis equipment. Likewise, although saliva is 98% water, glycoproteins such as mucins, as well as various protein enzymes, are present that may need to be removed prior to analysis. Several techniques are known for separating or removing protein, or otherwise extracting the water content from biological samples for analysis.
Conventional measurement technologies, which rely on using isotope ratio mass spectrometry, require conversion of the sample into a gas that can be isotopically characterized (e.g. H2, HD, D2, O2 and CO2). Thus, previous measurements of isotopic ratios of water in blood plasma or saliva typically involved extensive sample conditioning including distillation or direct sample conversion. Distillation is time consuming and onerous, and, because water of different isotopic compositions do not boil or evaporate at equal rates, if distillation is, to be used in preparation for quantitative analysis, then the sample must be distilled to completion so as to extract all of the water from the sample. Otherwise, the isotopes will fractionate and the measurement of the isotopic ratio will be inaccurate. Alternatively, the water in the sample can be converted to another species that is free of contaminants. For example, for 18O/16O measurements, the liquid sample can be equilibrated with CO2, and the CO2 can be measured using isotope ratio mass spectrometry to determine the 18O16O ratio of the water in the original liquid sample. Similarly, for 2H/1H measurements, the liquid sample can be reduced to H2 and HD using a hot zinc or chromium catalyst bed.
Recently, optical spectroscopy (e.g. tunable diode laser absorption spectrometry, cavity ringdown spectrometry, off-axis integrated cavity output spectrometry, and photoacoustic spectrometry) has been used to characterize the 2H/1H, 18O/16O and 17O/16O isotope ratios of water. In this technique, the water is measured directly without any sample conversion. This allows for direct characterization of slightly contaminated samples (e.g. seawater, groundwater, or urine) with minimal to no conditioning. However, these optical analyzers still exhibit difficulties in measuring blood plasma and saliva due to aggregation of the proteins inside the analyzer and sample handling equipment (syringes, evaporation blocks, etc.). This accumulation results in substantial measurement memory and large measurement inaccuracies.
Therefore, a simple and rapid method to remove these proteins without changing the isotope ratio of the liquid sample would enable optical analyzers to characterize the isotope ratios of bodily fluids in a simple, cost-effective and timely manner.
There are several methods of precipitating proteins, including “salting out” the protein, adding organic solvents to reduce the activity of water, adding organic polymers, acidifying the sample to form insoluble salts, and metal-induced affinity precipitation. Many of these techniques can be used to precipitate proteins for isotope studies.
“Salting out” the proteins is based on the principle that proteins are less soluble at higher salt concentrations. The added reagent or salt is often ammonium sulfate or sodium citrate, but other salts including both potassium and sodium sulfates and phosphates are also used as the precipitation reagent. The amount of protein precipitation (or the concentration of salt needed to be added) for a given choice of salt is indicated by the Hofmeister (or lyotropic) series: citrate3−>tartrate2−≈SO42−>HPO42−>F−>acetate−>HCO3−>Cl−>Br−>I−>NO3−>ClO3−>ClO4− for anions, and N(CH3)4+>Rb+>NH4+>K+>Na+>Li+>H+>Mg2+>Ca2+>Cu2+>Zn2+>Al3+ for cations, with precipitation tending to be increased for choices of anions and cations of the salt which occur earlier in the series. The sample may then be centrifuged to separate the proteins from the remaining liquid (mostly water) so as to facilitate subsequent measurements. This salting technique is used for fluid sample preparation in a variety of analytical contexts, including the measurement of NOx, glucose and creatine in blood plasma. However, some salts are inapplicable for isotope studies due to hydrogen-exchange with the liquid sample (e.g. ammonium salts, bicarbonates and other species with terminal exchangeable hydrogen). Moreover, high concentrations of salts are typically required, making it difficult for isotope analyzers to handle repeated samples due to their high salinity. Finally, even at high levels, “salting out” only removes at most 90% of the protein content of a sample, leaving the remaining 10% in solution and still confounding the sample processing.
Another technique, known as the Cohn process, adds ethanol or another organic solvent to the sample fluid so as to precipitate the proteins. This method is more commonly used to fractionate the proteins themselves, with different proteins precipitating out of a sample as the alcohol content increases and pH decreases. Other organic substances (e.g. methanol, acetone, ethylene glycol, trichloroacetic acid (TCA), and hydrazine) can be used in place of the ethanol, particularly where the interest is in the water rather than the protein content and one is not concerned whether or not the proteins are denatured. However, for most of these substances, the technique has the major drawback that the isotope ratio of the water will be changed by hydrogen exchange. Even if the isotope ratio of the substance(s) being added is known, control samples are used, and care is taken that all samples are treated identically, especially the amount of the added material, and computations are made to compensate for the change in isotope ratio, the accuracy of the result may still be adversely affected. Thus, this technique is limited to select solvents that do not exhibit hydrogen exchange (e.g. acetone, other ketones, dioxins and ethers).
A similar method involves adding an organic polymer to the solution to precipitate proteins. This soluble polymer, typically polyethylene glycol, usually has terminal, exchangeable hydrogens that alter the isotope ratio of the fluid. Therefore, this technique is typically unsuitable for isotope studies.
Another protein precipitation method involves adding acid to the solution to form insoluble salts with the amino groups in the protein. Again, this method is unsuitable for isotope studies due to hydrogen exchange of the terminal hydrogen in the added acid.
Yet another technique, flocculation, involving the addition of alginates, carrageenan, or tannins to separate the proteins, is little used. It also suffers the same drawback of changing the isotope ratio as the Cohn process and its variants.
The most promising method of precipitating proteins for isotope analysis involves metal-induced affinity precipitation. This technique involves adding a metal cation (e.g. Zn, Cu, Ca, Al) that forms protein cross-links and results in precipitation. The method is especially well-suited to isotope studies, because it involves a minimal quantity of the metal salt (thus minimizing salinity effects) and does not affect the isotope ratio of the sample (the counter-anion is typically sulfate or chloride, neither of which has exchangeable hydrogen).
Several research groups have used the aforementioned techniques to purify proteins (cf. R. K. Scopes, “Protein Purification: Principles and Practice”, 3rd ed., Springer, New York, 1994); however, very little work has focused on analysis of the remaining liquid, and there have been no known previous efforts involving characterization of the isotope ratios of that remaining liquid.
A. Ghesemi et al [“Protein Precipitation Methods Evaluated for Determination of Serum Nitric Oxide End Products by the Griess Assay”, J Medical Sciences Research, vol. 2, pp. 29-32 (Nov. 15, 2007)] disclose that deproteinization is necessary in NOx measurement of blood serum samples. Acetonitrile and zinc sulfate with ultrafiltration is the disclosed preferred method of deproteinization used by Ghesemi.
Zinc sulfate or other zinc salt together with methanol and ethylene glycol is the protein precipitation reagent disclosed in U.S. Pat. No. 5,135,875 of Meucci et al.
Zinc sulfate and barium hydroxide are used in the deproteinization method of both M. Somogyi [“Determination of Blood Sugar”, J Biological Chemistry, vol. 160, pp. 69-73 (1945)] and J. W. Kuan et al [“Determination of Plasma Glucose with Use of a Stirrer Containing Immobilized Glucose Dehydrogenase”, Clinical Chemistry, vol. 23, no. 6, pp. 1058-1061 (1977)] for the determination of plasma glucose (blood sugar).